This proposal has as its major goal the elucidation of the mechanisms by which the fidelity of DNA synthesis is maintained in mammalian cells. We will approach this problem by: 1) purifying to homogeneity the high molecular weight DNA polymerases of calf thymus: DNA polymerases Delta1 and Delta2, which contain a proofreading 3' to 5' exonuclease activity, and DNA polymerase Alpha, which is devoid of an exonuclease activity; 2) characterizing the physical and enzymatic properties of these polymerases, and investigating their possible interrelationships; 3) investigating the physiological roles of the polymerases by determining the relative levels of these enzymes in resting and dividing cells, and by determining the relative sensitivities of the polymerases to inhibitors of DNA replication; 4) determining the error frequency during DNA synthesis with these DNA polymerases using both synthetic polynucleotides and primed PhiX174 DNA as template/primers; 5) determining the effects of selective inhibition of the 3' to 5' exonuclease activity of DNA polymerases Delta1 and Delta2 on the fidelity of DNA synthesis; 6) investigating the interaction of the DNA polymerases with other replication proteins, and the effects of such interaction on the fidelity of DNA synthesis.